FT-IRRAS quantitative analysis of specific avidin adsorption on biotinylated Au surfaces

A new method has been developed for the preparation of amine-terminated surfaces with a highly cross-linked Si-O-Si network sub-layer on gold substrates. Biotinylation of the amine-terminated layers thus obtained can be easily accomplished by the use of an activated biotin reagent. Each step of the synthetic process was monitored by Fourier transformed infrared reflection adsorption spectroscopy (FT-IRRAS). Furthermore, the molecular recognition ability of the biotinylated surfaces was examined and quantified by the use of FT-IRRAS in combination with an alkyne dicobalt hexacarbonyl probe coupled with netravidin. The specific binding capacity of the avidin on the biotinylated surfaces was measured to be approximately 3.5 pmol/cm2. FT-IRRAS was shown to be very sensitive to the detection of avidin with a limit of 0.1pmol/cm2. The non-specific adsorption of the avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to a solution of biotin-saturated avidin. The results in this case indicate that both the biotinylated and non-biotinylated (i.e., amine-terminated) surfaces synthesized in this study are resistant to non-specific protein adsorption, exhibiting a selectivity towards specific binding of 97%.